Solid phase extraction is a powerful sample preparation tool to perform extraction, fractionation, pre-concentration as well as clean up of undesired compounds from analytical samples. For example, the technique has found success in monitoring drinking water by extraction of organics from the water followed by high pressure liquid chromatography separation and mass spectrometry (MS) detection to determine the identity and concentration of pollutants. Proteins and nucleic acid materials are frequently isolated from biological samples by passing them through a packed column and cartridge containing a solid phase where the molecules of interest are adsorbed. After the sample has passed through the column and the sample molecules have been adsorbed, a solvent is used to desorb the molecules of interest and form a concentrated solution. It is particularly important to be able to purify and concentrate non-polynucleotide biomolecules such as polypeptides and polysaccharides, since these molecules are not amenable to the types of amplification techniques routinely used with nucleic acids.
Proteomics has recently been recognized as the comprehensive identification and the analysis of proteins within cells. The identification and analysis of proteins could potentially lead to technologies that could artificially control mechanisms of cell proliferation differentiation, growth and aging. Information obtained from the analysis of proteins is particularly advantageous compared to the analysis of deoxyribonucleoteic acid (DNA) because it is possible to gain information regarding post-translational modifications (PTMs).
A major challenge for proteomics is the study of low-abundance proteins. Many proteins and peptides are only expressed at extremely low levels, and in the presence of a vast excess of contaminating proteins and other cellular constituents. In some eukaryotic cells, the amounts of the most abundant proteins can be 106-fold greater than those of the low-abundant proteins. Many important classes of proteins (that may be important drug targets) such as transcription factors, protein kinases, and regulatory proteins are low-copy proteins. These low-copy proteins will not be observed in the analysis of crude cell lysates without some prior purification. Therefore, new methods must be devised for the sub-proteome isolation.
Functional proteome characterization involves the challenging task of identifying species of interest from among many thousands of proteins, each potentially altered by hundreds of possible post-translational modifications. Additionally, the living organisms often exhibit large dynamic range of protein expression levels, ranging from estimated values of 104 in yeast to 109-1012 in plasma. As a result of this extreme complexity, proteomics studies often use various fractionation methodologies to focus on only a subset of the overall protein complement. For example, numerous fractionation schemes based on the presence of a particular chemical moiety such as native amino acid side-chain functionality or biologically important PTMs have been described. Often, these affinity methods are specific for a particular functionality, such as immobilized metal affinity chromatography for the enrichment of phosphorylated peptides or various lectins for the enrichment of specific glycosylated species. Any viable proteomics fractionation and pre-concentration methodology must enable both the efficient isolation of a desired subset from the remainder of the sample, as well as the subsequent efficient recovery and analysis of that subset. In addition the method has to be capable of maintaining the protein functionality i.e. the enzyme activity. The developed methods must be easily integrated into the miniaturized systems.
It is often necessary to purify and concentrate a protein sample of interest prior to performing analytical techniques such mass spectrometry, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), X-ray crystallography and the like. These techniques typically only require a small volume of sample, but it must be presented at a sufficiently high concentration and interfering contaminants should be removed. Hence, there is a need for sample preparation methods that permit the manipulation and processing of small sample volumes with minimal sample loss.
Methods and reagents for performing solid phase extractions in open channels, such as open capillaries, are described in co-pending U.S. patent application Ser. No. 10/434,713. The instant disclosure follows up on that application, providing in some instances more specific and detailed teaching for performing open channel solid phase extractions. These methods, and the related devices and reagents, will be of particular interest to the life scientist, since they provide a powerful technology for purifying, concentrating and analyzing biomolecules and other analytes of interest. However, the methods, devices and reagents are not limited to use in the biological sciences, and can find wide application in a variety of preparative and analytical contexts.